Pharmaceutical composition for use in the treatment or prevention of osteoarticular diseases

ABSTRACT

A pharmaceutical composition for the acute and/or chronic treatment or prevention of osteoarticular diseases includes an adequate pharmaceutical carrier or diluent, a polysaccharide and/or a glycosaminoglycan, an anti-inflammatory agent and stem cells.

This application is a Continuation of U.S. patent application Ser. No.12/867,719, filed Oct. 20, 2010, which is a National Stage Applicationof International Patent Application No. PCT/EP2009/051816, filed Feb.16, 2009, which claims benefit of Ser. No. 08/158,284.3, filed Jun. 13,2008 in the EPO, and also of Ser. No. 08/101,683.4, filed Feb. 15, 2008in the EPO which applications are incorporated herein by reference. Tothe extent appropriate, a claim of priority is made to each of the abovedisclosed applications.

FIELD OF THE INVENTION

The present invention is in the pharmaceutical field and is related to anew pharmaceutical composition for use in the treatment or theprevention of the acute and/or chronic osteoarticular diseases.

BACKGROUND OF THE INVENTION

Osteoarthritis, the most common form of arthritis, is a diseasecharacterised by a slow degenerative processes in the articularcartilage, subchondral bone associated with marginal osteophyteformation, and low grade inflammation. Osteoarthritis is believed toaffect 15% of the population in its chronic form. Of those, one-quarterare severely disabled. Most cases of osteoarthritis have no known causeand are referred to as primary osteoarthritis. When the cause of theosteoarthritis is known, the condition is referred to as secondaryosteoarthritis. Secondary osteoarthritis is caused by another disease orcondition. Conditions that can lead to secondary osteoarthritis includerepeated trauma or surgery to the joint structures, abnormal joints atbirth (congenital abnormalities), gout, diabetes, and other hormonedisorders. Other forms of arthritis are systemic illnesses, such asrheumatoid arthritis and systemic lupus erythematosus (SLE).

Osteoarthritis involves mainly the hips, knees, spine, and theinterphalangeal joints. The most common symptom of osteoarthritis ispain in the affected joint(s) after repetitive use. Joint pain isusually worse later in the day. There can be swelling, warmth, andcreaking of the affected joints. Pain and stiffness of the joints canalso occur after long periods of inactivity. In severe osteoarthritis,complete loss of cartilage cushion causes friction between bones,causing pain at rest or pain with limited motion.

Osteoarthritis is characterized by a slow degradation of cartilage overseveral years. In normal cartilage, a delicate balance exists betweenmatrix synthesis and degradation; in osteoarthritis, however, cartilagedegradation exceeds synthesis. The balance between synthesis anddegradation is affected by age and is regulated by several factorsproduced by the synovium and chondrocytes, including cytokines, growthfactors, aggrecanases, and matrix metalloproteinases. In addition towater, the extracellular matrix is composed of proteoglycans, made up ofglycosaminoglycans attached to a backbone made of hyaluronic acid,entrapped within a collagenous framework or fibrillary matrix. Asignificant proteoglycal in articular cartilage is aggrecan, which bindsto hyaluronic acid and helps provide the compressibility and elasticityof cartilage. Aggrecan is cleaved by aggrecanases, leading to itsdegradation and to subsequent erosion of cartilage. The loss of aggrecanfrom the cartilage matrix is one of the first pathophysiological changesobserved in OA.

Cytokines produced by the synovium and chondrocytes, especially IL-1betaand Tumor Necrosis Factor alpha (TNF-alpha), are also key players in thedegradation of cartilage. IL-1beta is spontaneously released fromcartilage of osteoarthritis but not normal cartilage. Both IL-1beta andTNF-alpha stimulate their own production and the production of othercytokines (e.g., IL-8, IL-6, and leukotriene inhibitory factor),proteases, and prostaglandin E₂ (PGE₂). Synthesis of the inflammatorycytokines IL-1beta and TNF-alpha and expression of their receptors areenhanced in osteoarthritis. Both cytokines have been shown to potentlyinduce degradation of cartilage in vitro. Other proinflammatorycytokines overexpressed in osteoarthritis include IL-6, IL-8, IL-11, andIL-17, as well as leukotriene inhibitory factor.

The extracellular matrix (ECM) composing the cartilage is degraded bylocally produced matrix metalloproteinases. Proinflammatory cytokines,including IL-1beta, TNF-alpha, IL-17, and IL-18, increase synthesis ofmatrix metalloproteinases, decrease matrix metalloproteinase enzymeinhibitors, and decrease extracellular matrix synthesis.

In an attempt to reverse the breakdown of the extracellular matrix,chondrocytes increase synthesis of matrix components includingproteoglycans. Even though this activity increases, a net loss ofproteoglycans in the upper cartilage layer is seen. Elevatedanti-inflammatory cytokines found in the synovial fluid ofosteoarthritis include IL-4, IL-10, and IL-13. Their role is to reduceproduction of IL-1beta, TNF-alpha, and matrix metalloproteinases, andinhibit prostaglandin release. Local production of growth anddifferentiation factors such as insulin-like growth factor 1,transforming growth factors, fibroblastic growth factors, and bonemorphogenetic proteins also stimulate matrix synthesis.

STATE OF THE ART

Currently available pharmacological therapies target palliation of painand include analgesics (i.e. acetaminophen, cyclooxygenase-2-specificinhibitors, non-selective nonsteroidal anti-inflammatory drugs,tramadol, opioids). However, the clinical presentation of osteoarthritisis usually monoarticular or oligoarticular with fluctuations inintensity and localisation over time. It is therefore logical toconsider local therapeutic modalities in order to avoid untowardsystemic effects. Several compounds have been used intra-articularly(e.g., glucocorticoids, hyaluronic acid) or topically (e.g., capsaicin,methylsalicylate). However, the benefit of intra-articularglucocorticoides lasts only a few days (Barron, M. C., 2007, J. Am.Osteopath., 107, ES21-27).

Polysaccharides form a class of materials whose recognition of thepotential utility is growing. Apart from their biological activity, oneof the most important properties of polysaccharides in general is theirability to form hydrogels. Hydrogel formation can occur by a number ofmechanisms and is strongly influenced by the types of monosaccharideinvolved, as well as the presence and nature of substituent groups.Polysaccharide gel formation is generally of two types: hydrogen bondedand ionic. Hydrogen-bonded gels are typical of molecules such as agarose(thermal gellation) and chitosan (pH-dependent gellation), whereasionically bonded gels are characteristic of alginates and carrageenans.

Proteoglycans are one of the major macromolecules found in articularcartilage. These molecules consist of a core protein and covalentlyattached glycosaminoglycans (GAG) chains. The GAGs are long, unbranchedheteropolysaccharides, consisting of repeated disaccharide units, withthe general structure: Muronic acid amino sugar. The cartilage-specificGAGs include chondroitin 4-sulfate (glucuronic acid andN-acetyl-galactosamine with an SO₄ on the 4-carbon position),chondroitin 6-sulfate (glucuronic acid and N-acetyl-galactosamine withan SO₄ on the 6-carbon position) and keratan sulfate (galactose andN-acetyl-glucosamine with an SO₄ on the 6-carbon position).

These molecules are capable of forming hydrogel complexes withoppositely charged ionic polymers, particularly the cationicpolysaccharide chitosan. This interaction may form the basis of a newmaterials approach to cartilage tissue engineering. The other importantcartilage GAG is hyaluronic acid (glucuronic acid andN-acetyl-glucosamine). This molecule is one of the major components insynovial fluid. Hyaluronic acid molecules are also present in cartilagematrix as the backbone structure in proteoglycan aggregates. In general,hyaluronic acid plays a major role as an organizer of the extracellularmatrix. Purified hyaluronic acid is employed as a structural biomaterialbecause of its high molecular weight and gel forming ability. Theproperties of the molecule may be broadly altered by chemicalmodification. For example, partial esterification of the carboxyl groupsreduces the water solubility of the polymer and increases its viscosity.Extensive esterification generates materials that form water-insolublefilms or swellable gels. Ethyl and benzyl esterified hyaluronatemembranes have excellent healing responses and biodegradabilityproperties. The fully esterified membranes have in vivo lifetimes ofseveral months, whereas the partially esterified forms have been shownto degrade within a few weeks.

Hyaluronic acid is responsible for the viscoelastic quality of synovialfluid that acts as both a lubricant and shock absorber. In synovialfluid, hyaluronic acid coats the surface of the articular cartilage andshares space deeper in the cartilage among collagen fibrils and sulfatedproteoglycans. It protects the cartilage and blocks the loss ofproteoglycans from the cartilage matrix into the synovial space,maintaining the normal cartilage matrix. In synovial fluid from kneejoints in osteoarthritis, concentrations of hyaluronic acid,glycosaminoglycans, and keratan sulfate are lower than in synovial fluidfrom normal knee joints. Additionally, experiments using rabbit synovialcells showed that the proinflammatory cytokines IL-1 and TNF-alphastimulate the expression of hyaluronic acid synthetase, which maycontribute to the fragmentation of hyaluronic acid under inflammatoryconditions. Exogenous hyaluronic acid may facilitate the production ofnewly synthesized hyaluronic acid. Hyaluronic acid and derivatives havebeen used as therapeutic aids in the treatment of osteoarthritis as ameans of improving lubrication of articulating surfaces and thusreducing joint pain. Several in vitro culture studies have alsodemonstrated that hyaluronic acid has a beneficial effect by inhibitingchondrocytic chondrolysis mediated by fibronectin fragment. Hyaluronicacid has also been shown to have anti-inflammatory effects, as well asinhibitory effects on prostaglandin synthesis, and proteoglycan releaseand degradation.

Chitosan, a partially de-acetylated derivative of chitin, found inarthropod exoskeletons is another proteoglycan, a linear polysaccharideconsisting of beta(1-4) linked D-glucosamine residues with a variablenumber of randomly located N-acetyl-glucosamine groups. It thus sharessome characteristics with various GAGs and hyaluronic acid present inarticular cartilage, Depending on the source and preparation procedure,chitosan's average molecular weight may range from about 50 to about1000 kDa. Commercially available preparations have degrees ofdeacetylation ranging from about 50 to about 90%. Chitosan is asemi-crystalline polymer and the degree of crystallinity is a functionof the degree of deacetylation. Crystallinity is maximum for both chitin(i.e. 0% deacetylated) and fully deacetylated (i.e. 100%) chitosan.Minimum crystallinity is achieved at intermediate degrees ofdeacetylation.

Much of the potential of chitosan as a biomaterial stems from itscationic nature and high charge density. The charge density allowschitosan to form insoluble ionic complexes or complex coacervates with awide variety of water-soluble anionic polymers.

In fact, chitosan oligosaccharides have been shown to have a stimulatoryeffect on macrophages, and the effect has been linked to the acetylatedresidues. Furthermore, both chitosan and its parent molecule, chitin,have been shown to exert chemoattractive effects on neutrophils in vitroand in vivo. In vivo, chitosan is degraded by enzymatic hydrolysis. Themechanical properties of chitosan scaffolds are mainly dependent on thepore sizes and pore orientations.

Hyaluronic acid, a glycosaminoglycan, is widely used for the treatmentof osteoarthritis of the knee. A survey of 2 general practices in theUnited Kingdom showed that about 15% of patients with osteoarthritisreceived intra-articular treatment with hyaluronic acid preparations.Because of its viscoelastic quality, it may replace synovial fluid.Furthermore, it may reduce the perception of pain. Beneficial molecularand cellular effects have also been reported. Hyaluronic acid isfrequently applied by intra-articular injection, but the evidenceconcerning its clinical relevance is conflicting. State-of-the-artsystematic reviews and meta-analyses were published recently, and theirauthors concluded that intra-articular hyaluronic acid, at best, has asmall effect, a clinically meaningful effect meaning an improvement ofat least 15 mm on the visual analog scale of pain (Bellamy et al, 2006;Cochrane Database Syst Rev. 2006 Apr. 19; (2):CD005321). These data formthe basis for the use of intra-articular administration of hyaluronicacid in patients with osteoarthritis. The benefits are sometimes noticedonly one year after injections and, in some experiments, injections mustbe performed three to five-times a week (Barron, M. C., 2007, J. Am.Osteopath., 107, ES21-27).

Alpha-2-adrenergic receptor ligands especially agonists are drugscommonly used in medical practice as antihypertensive substance and inclinical anesthesiology as component of general and locoregionalanesthesia and analgesia. They produce anxiolysis, analgesia, sedation,anesthetic sparing effects and peri-operative hemodynamic stabilizingeffects. Negative neurotoxicity studies allow their use (mainlyclonidine) by systemic and perimedullar routes and for peripheral nerveblocks. Among the clinically available alpha-2-adrenoreceptors agonists,clonidine remains widely used: the substance is devoid of neurotoxicityand displays less side effects (i.e. hypotension and sedation) than themore potent and also more alpha-2-adrenergic receptor selective agonist,dexmedetomidine. Clonidine, a potent alpha-2-adrenergic receptorspartial agonist, was used primarily for the treatment of hypertension.This drug stimulates alpha-2-adrenergic receptors in the vasomotorcenters, causing a reduction of sympathetic outflow from the centralnervous system. Both cardiac output and peripheral resistance arereduced resulting in a decrease in blood pressure. Higher concentrationscause a vasoconstriction by activation of postsynaptic receptors invascular smooth muscle. However, the significant advantages of the drugare counter balanced by side effects including dryness of the mouth,sedation and dizziness. Furthermore, other activities of these compoundssuch as anti-inflammatory effect have never been reported by oraladministration.

Besides the well-known analgesic effects of spinally administeredalpha-2-adrenergic (alpha-2-adrenoceptors) agonists, their peripheraluse has been commonly reported in acute pain conditions. Forperi-operative analgesic techniques, clonidine is added to localanaesthetic in peritroncular nerve blocks to enhance potency andduration of analgesia. ZOUHER A et al.; Paediatr Anaesth. 2005 November;15(11):964-70; Luiz-Cleber P. et al.; Anesth Analg. 2005 September;101(3):807-11, Murphy D R “A non-surgical approach to low back pain”Med. Health R. I., 2000 April; 83(4): 104-7). Further, intra-articularinjection of clonidine and its adjunction to local anaesthetic solutionfor intravenous regional anaesthesia have also displayedanti-nociceptive effect S. Armand et al; Br J Anaesth. 1998 August;81(2):126-34; Gentili M, et al. Pain 1996; 64: 593-596; Reuben S, et al.Anesthesiol. 1999; 91: 654-658. However, the effect is here designed notto last for days or weeks.

Alpha-2-adrenoceptors agonists are known to block the tissue contentincrease of pro-inflammatory cytokines, such as TNF-alpha and IL-1betaand increase the tissue content of anti-inflammatory cytokine TGF beta.This has been shown in inflammatory neuropathic pain model by partialligation of sciatic nerve, by applying locally alpha 2 adrenergicreceptor agonists by peripheral nerve block.

BRIEF DESCRIPTION OF THE DRAWINGS

Alpha FIG. 1 shows the attachment of peripheral blood mononuclear cells(PBMCs) on the surface of mesenchymal stromal cells (MSCs).

Alpha FIG. 2 shows Alcian blue staining of pellets obtained by culturingmesenchymal stromal cells (MSCs) in chondrogenic medium treated withp-aminoclonidine (A2A), hyaluronic acid (HA) or A2A and HA at lowmagnification power (4×) and high magnification power (40×).

SUMMARY OF THE INVENTION

The invention is related to a new (intra-articular) pharmaceuticalcomposition for use in the treatment and/or the prevention of acute orchronic osteoarticular diseases and acute or chronic osteoarticularsymptoms especially osteoarthritis, this composition comprising

-   -   possibly an adequate pharmaceutical carrier or diluent    -   a polysaccharide and/or a glycosaminoglycan, preferably a        glycosaminoglycan (including proteoglycan),    -   stem cells (differentiated or not), but with the provisio that        these stem cells are not human embryonic stem cells, having        potential anti-inflammatory properties and    -   a sufficient amount of an anti-inflammatory agent (compound).

In the pharmaceutical composition of the invention the polysaccharide orthe glycosaminoglycan (including the proteoglycan) are present as a filmor a matrix, preferably in the form of a paste or a gel, more preferablyan hydrogel with a sufficient amount of an aqueous solvent.

Preferably, the anti-inflammatory agent (compound) is selected from thegroup consisting of a steroidal (prednisolone, dexamethasone,betamethasone, triamcinolone . . . ), a non-steroidal anti-inflammatorycompound (ibuprofen, diclofenac, naproxen, cox-2 inhibitors etc. . . .), a disease modifying antirheumatic drug (DMARD such as methotrexate,leflunomide, etc. . . . ), an alpha 2 adrenergic receptor agonist, ananti-CD20 agent, an anti-cytokine agent (anti-IL1, anti-IL6, anti-IL17),an anti-TNF agent (infliximab, etanercerpt, adalimumab, rituximab, etc)or a mixture thereof.

Advantageously, the anti-inflammatory compound is a compound interactingwith the alpha-2-adrenergic receptor, preferably an alpha-2-adrenergicreceptor agonist.

The alpha-2-adrenergic receptor agonist is selected from the groupconsisting of clonidine, p-aminoclonidine, tiamenidine, 5-bromo-6-(2imidazolidine-2-ylamino) quinoxaline, dexmedetomidine, detomidine,medetomidine, alphamethyldopa, oxymetazonline, brimonidine, tizanidine,mivazerol, lofexidine, xylazine, guanfacine, guanclofine, guanoxabenz,or a derivative or structural analogue thereof, alpha-methyinorepherine,azepexole, indoramin, 6-allyl-2-amino-5,6,7,8-tetrahydro4H-thiazolo[4,5-d]azepine diHCl or a compound identified in the table 1.

In the composition according to the invention the polysaccharide-basedhydrogel and the glycosaminoglycan are not covalently linked or arecovalently linked.

Preferably, the glycosaminoglycan is selected from the group consistingof hyaluronic acid with low (<900 kDa) or high (>900 kDa) molecularmass.

In the composition of the invention, the hyaluronic acid and the alpha 2adrenergic receptor agonist are not covalently linked or are covalentlylinked.

In the composition according to the invention the glycosaminoglycan isselected from the group consisting of proteoglycan, chondroitin sulfate,keratin sulphate, hyaluronic acid, (including their derivative),chitosan, a chitosan or chitin derivative, or a mixture thereof.

Advantageoulsy, the non human embryonic stem cells of the compositionare selected from the group consisting of (cells obtained from) bonemarrow or adipose tissue or bone tissue or joint tissue, a mix ofhematopoietic and mesenchymal stromal cells, (expanded or not),mesenchymal stromal cells, osteoblastic cells, chondrocytic cells or acombination thereof.

The inventors have discovered that the pharmaceutical compositionaccording to the invention is suitable for a treatment or prevention ofacute and/or chronic osteoarticular diseases and associated symptoms(especially osteoarticular pain, mobility or function), of inflammatoryorigin, such as osteoarthritis, degenerative arthristis, degenerativearthritis, gonarthrosis, coxarthrosis, and other inflammatory generalconditions in which joints are involved, such as autoimmune diseases,especially rheumatoid arthritis and systemic lupus erythematosus (SLE)spondyloarthropathies, polymyalgia rheumatica, ankylosing spondylitis,Reiter's Syndrome, psoriatic arthropathy, enteropathic arthritis(related to inflammatory bowel disease, such as haemorrhagic colitis andCrohn's disease), neuropathic arthropathy, acute rheumatic fever, gout,chondrocalcinosis, calcium hydroxyapatite crystal deposition disease,Lyme disease and all other degenerative joint diseases.

The composition of the invention is also suitable for obtaining anefficient proliferation of stem cells therefore, for improvingregenerative or damaged tissues. This effect upon regeneration oftreated tissues is advantageously induced by the synergic effect of theactive compounds present in the composition of the invention.

Therefore, the composition according to the invention is characterizedby an anti-inflammatory activity, a tissue or cell regenerative effect,and an advantageous efficient treatment and/or prevention of acuteand/or chronic osteoarticular diseases and/or symptoms. These effectscould be combined in the composition according to the invention.

According to a preferred embodiment of the present invention thecomposition presents a preferred formulation, preferably being aninjectable solution for delivering an efficient amount (therapeuticdose) of the active compound present in the composition to a mammalsubject including a human patient, especially in the knees, the hips andthe spine of the subject.

Preferably, this injectable solution comprises from about 0.1 mg toabout 100 mg/kg preferably from about mg to about 10 mg/kg of bodyweight of this polysaccharide and/or glycosaminoglycan, preferably inthe form of a polysaccharide-based hydrogel and may comprise from about0.1 mg to about 100 mg/kg of patient body weight preferably from about0.1 mg to about 0.8 mg/kg of patient body weight of an anti-inflammatorycompound, being preferably a compound activating the alpha-2-adrenergicreceptor, preferably an alpha-2-adrenergic receptor agonist.

The injectable solution is adequate for intra-articular administration(percutaneous injection) in a joint of a mammal subject, preferably of ahuman patient.

This formulation is also adequate for local administration (percutaneousinjection in/or in the vicinity of an inflamed joint of a mammalsubject, preferably of a human patient), local administration injectionthat does concern the epidermis, the muscle or any deep organs.

Another aspect of the present invention is related to the use of thepharmaceutical composition for the manufacture of a medicament in thetreatment and/or the prevention of these mentioned diseases and/orsymptoms (pain) induced by these diseases.

The present invention is related to the surprising discovery that thisintra-articular administration to a mammal subject, particularly a humanpatient of this pharmaceutical composition results in an improvement ofsymptoms associated with osteoarticular diseases, such as a relief ofosteoarticular pain, an improvement of joint mobility and/or function, adecrease in articular accumulation of inflammatory liquid, induced bythe above mentioned diseases or pathologies.

The present invention is also related to the surprising discovery thatthe intra-articular administration to a mammal subject, particularly ahuman patient of the pharmaceutical composition according to theinvention results in both a reduction of inflammation, and of jointdegeneration, resulting in disease improvement and joint regenerativeeffects, combined with the above effects in the improvement of symptomsassociated with osteoarticular diseases. These effects are obtained bythe synergic combination of the elements present in the composition.

The present invention is also related to the surprising discovery thatthe intra-articular administration to a mammal subject, particularly ahuman patient of the pharmaceutical composition according to theinvention results also in a shortening of the time to onset of thetherapeutic activity of the first component, an increase in the durationof action of the first component.

The composition and method of the present invention also allows adecrease of the number of required administrations to obtain a desiredefficacy and allows for a faster onset of action.

The invention is also directed to a method for a treatment and/or aprevention (prophylaxis) of a condition selected from the groupconsisting of the above mentioned diseases or symptoms through anadministration to a mammal subject, preferably a human patient of asufficient amount of the pharmaceutical composition of the invention,through an intra-articular administration, particularly in the knees,the hips and the spine or any joint (e.g., interphalangeal) of themammal subject; preferably the human patient.

A last aspect of the present invention is related to a kit of partscomprising one or more vials with the elements (carrier/diluent,polysaccharide, glycosaminoglycan, anti-inflammatory agent (orcompound), stem cells or a combination thereof) of the composition ofthe invention and a device for delivering these elements (simultaneouslyor successively) or this composition to an inflamed joint of a mammalsubject, preferably a human patient suffering from the above mentionedosteoarticular diseases or symptoms (in particular pain) and having

-   -   reservoir means for storing this pharmaceutical composition,    -   piston means movable along the longitudinal axis of the        reservoir for dispensing this pharmaceutical composition and,    -   a hollow needle mounted on said reservoir means for delivering        this pharmaceutical composition to the peripheral nerve of the        mammal subject.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying example.

DETAILED DESCRIPTION OF THE INVENTION

The term “intra-articular” as used herein refers to a percutaneousinjection of a joint with the pharmaceutical composition of theinvention.

The term “local administration” as used herein refers to a percutaneousinjection in or in the vicinity of an inflamed joint. Localadministration injection thus concerns the epidermis, the dermis, themuscle, or any deep organ.

The main advantages to local administration are to selectively restrictthe analgesic effect to the injured areas. Furthermore, the localadministration allows for high local concentration levels with little orno systemic release. The local administration and the intra-articularinjection of hyaluronic acid is a recognised treatment of the abovementioned conditions.

The local administration and the intra-articular injection of theanti-inflammatory compound and stem cells are easily realizable andprovide long lasting effect in combination with proteoglycans. Inconsequence, the problems related to placement of an invasive drugdelivery system and the problems of bothersome side-effects, due tosystemic release, can be strongly minimized. Health-related quality oflife, patient satisfaction and economic assessment might be improvedwith such a treatment, especially in chronic conditions.

Preferably, the administrated proteoglycan is selected from the groupconsisting of a polysaccharide-based hydrogel or a glycosaminoglycan,for example hyaluronic acid or a salt thereof or an ester of hyaluronicacid with an alcohol of the aliphatic, heterocyclic or cycloaliphaticseries, or a sulphated form of hyaluronic acid or combination of agentscontaining hyaluronic acid. Suitable dosages of a polysaccharide-basedhydrogel or a glycosaminoglycan or a derivative thereof will typicallybe from about 0.1 mg to about 100 mg/kg body weight per day or fromabout 0.5 mg to about 10 mg/kg body weight per day more preferably fromabout 2 mg to about 8 mg by body weight per day.

Advantageously, the administrated stem cells are from bone marrow,adipose tissue, bone tissue or joint tissue, composed of a mix ofhematopoietic and mesenchymal stromal cells, of mesenchymal stromalcells—expanded or not, of osteoblastic cells or chondrocytic cells orany combination thereof.

According to a first embodiment, the administrated stem cells areosteoblastic cells, or preferably a cell population selected from thegroup consisting of osteoprogenitors, pre-osteoblasts and osteoblasts.

According to the second embodiment, the administrated stem cells arechondrocytic cells, or even preferably a cells population selected fromthe group consisting of chondroprogenitors, pre-chondrocytes andchondrocytes.

In a preferred embodiment, the alpha 2 adrenergic receptor agonist maybe clonidine, p-aminoclonidine, tiamenidine, 5-bromo-6-(2imidazolidine-2-ylamino) quinoxaline, dexmedetomidine, detomidine,medetomidine, alphamethyldopa, oxymetazonline, brimonidine, tizanidine,mivazerol, lofexidine, xylazine, guanabenz, guanfacine, guanclofine,guanoxabenz, or a derivative or structural analogue thereof,alpha-methyinorepherine, azepexole, indoramin,6-allyl-2-amino-5,6,7,8-tetrahydro4H-thiazolo [4,5-d]azepine diHClanalogs thereof or a compound selected from the table 1 and analogsthereof.

TABLE 1 Classification of the alpha-2-adrenergic receptor agonistsguanidines agmatine betanidine biguanides cimetidine creatine gabexateguanethidine guanfacine guanidine impromidine iodo-3 benzylguanidinemethylguanidine mitoguazone nitrosoguanidines pinacidil robenidinesulfaguanidine zanamivir imidazoles4-(3-butoxy-4-méthoxybenzyl)imidazolidin-2-one acide urocaniqueamino-imidazole carboxamide antazoline biotine bis(4-methyl-1-homopiperazinylthiocarbonyl)disulfide carbimazole cimetidine clotrimazolecreatinine dacarbazine dexmedetomidine econazole enoximone ethymizoletomidate fadrozole fluspirilène histamine histidinol idazoxanimidazolidines imidazolines clonidine tolazoline impromidine levamisolelosartane medetomidine miconazole naphazoline niridazole nitroimidazolesondansetron oxymetazoline phentolamine tetramisole thiamazoletrimetaphan Derivatives of clonidine 2,6-dimethylclonidine4-azidoclonidine 4-carboxyclonidine-methyl 3,5-dichlorotyrosine4-hydroxyclonidine 4-iodoclonidine alinidine apraclonidinechlorethylclonidine clonidine 4-isothiocyanate clonidine4-methylisothiocyanate clonidine receptor clonidine-displacing substancehydroxyphenacetyl aminoclonidine N,N′-dimethylclonidine

The active compounds used in accordance with the invention are known.Pharmaceutical preparations containing hyaluronic acid are commerciallyavailable as are clonidine and other alpha 2 adrenergic receptoragonists. The compounds can be manufactured in a known manneressentially in accordance with the processes described in the prior art.

The term “clonidine” as used herein refers toN-(2,6-dichlorophenyl)-4,5-dihydro-1H-imidazol-2-amine and includes thepharmaceutically acceptable salts thereof, e.g., salts with inorganicacids, such as hydrohalic acids, or with organic acids, for examplelower aliphatic monocarboxylic or dicarboxylic acids such as aceticacid, fumaric acid or tartaric acid or aromatic carboxylic acids such assalicylic acid are also suitable.

Clonidine is employed in a therapeutically effective amount. The presentinvention also encompasses the use of alpha 2 adrenergic receptoragonist for the manufacture of injectable formulations for a delivery toa joint or a close region thereof, a therapeutic dose of said agonist byintra-articular injection, wherein said solution comprises from about 3μg to about 1500 μg of said alpha 2 adrenergic receptor agonist.Preferably, said formulation comprises from about 30 μg to about 500 μgof said alpha 2 adrenergic receptor agonist. More preferably saidsolution comprises from 50 μg to 350 μg of said alpha 2 adrenergicreceptor agonist. In a preferred embodiment, the alpha 2 adrenergicreceptor agonist is clonidine. The actual concentration of clonidine mayvary, depending on the nature and degree of the pain syndromes beingtreated and whether the drug is being administered for therapeutic orprophylactic purposes.

It is hypothesized that based on their anti-inflammatory properties,inhibition of pro-inflammatory cytokines TNF-alpha and IL1-beta and theincrease of anti-inflammatory cytokines such as TGF beta, alpha 2receptor agonists in injectable administration will have applications inosteoarticular inflammatory conditions and in diseases where there areosteoarticular inflammatory conditions, such as those above mentioned.

According to the method and the formulation of the invention, aninjection of the combination of hyaluronic acid and an alpha 2adrenergic receptor agonist form induces a long lasting pain relief,both in human case and in an animal model of osteoarticular pain. Themethod is safe, devoid of major drug's side effects and allows for acuteas well as chronic treatment and or prophylaxis without the use of tooinvasive technique.

Preservatives may be incorporated in an amount effective for inhibitinggrowth of microbes, such as bacteria, yeast and molds, in thecomposition. Any conventional preservative against microbialcontamination of the product can be employed so long as it ispharmaceutically acceptable and is unreactive with clonidine. Preferredpreservatives are antimicrobial aromatic alcohols, such as benzylalcohol, phenoxyethanol, phenethyl alcohol, and the like, and esters ofparahydroxybenzoic acid commonly referred to as paraben compounds, suchas methyl, ethyl, propyl, and butyl esters of parahydroxybenzoic acidand the like and mixtures thereof, but are not limited thereto.Particularly preferred are benzyl alcohol and phenoxyethanol.

Optionally, anaesthetic agent, such as lidocaine and the like, can beincluded. For administration according to the invention the activequantities of the compounds that alleviate neuropathic pain can becontained together with customary pharmaceutical excipients and/oradditives in solid or liquid pharmaceutical formulations.

Liquid preparations such as solutions, suspensions or emulsions of theactive ingredients, prepared by dissolving or dispersing the compounds,can contain the usual diluents such as water, oil and/or suspending aidssuch as polyethylene glycols and optional pharmaceutical adjuvants, in acarrier, such as, for example, aqueous saline, aqueous dextrose,glycerol, or ethanol, to form a solution or suspension and such like.Further additives such as preservatives, flavouring agents and such likemay also be added.

Example

The effects of the combination of alpha 2 adrenoreceptor agonists (e.g.,clonidine), of a glycosaminoglycan (e.g., hyaluronic acid) and of stemcells were tested in an inflammation model with stimulated PeripheralBlood Mononuclear Cells.

PBMCs (Peripheral blood mononuclear cells) from heparinized venous bloodof healthy volunteers were isolated by Ficoll-gradient centrifugation.MNC (Mononuclear cells) were washed three times in PBS and resuspendedin RPMI-1640 medium supplemented with 100 U/mL penicillin, 100 μg/mLstreptomycin, and 10% heat-inactivated FBS (Fetal Bovine Serum). Cellswere seeded at 100.000 cells in 96-well plate in a total volume of 200μL/well.

PBMC, at 100.000 cells/200 μL, were plated in 96-well microtiter platesand stimulated or not with 10 μg/mL phytohemagglutinin (PHA). Effects ofhyaluronic acid “HA” (200 μg/mL) and p-aminoclonidine “A2A” (5 μM) weretested on PBMC.

MSC (Mesenchymal stromal cells) were derived from 20 to 60 mL ofheparinized bone marrow (BM) obtained from the iliac crest of healthyvolunteers. BM was mixed with phosphate-buffered saline (PBS, 2v:v) andlayered on density gradient Ficoll solution. After centrifugation,mononuclear cells were harvested from the interface and washed twice inPBS. Cells were resuspended in standard MEM medium supplemented with 15%FBS. The cells were plated at 2×10⁶ cells/75 cm2 flasks and maintainedin a 37° C. humidified atmosphere containing 5% CO2. Cells were allowedto attach for 24 hours prior an initial medium change. Cells weredetached at day 10 using trypsin-EDTA solution for 1-5 min at 37° C.Then, cells were irradiated (10 minutes at 2200 Ci) before seeding in96-well for proliferative assays at 12.500 cells/well to obtain a ratio1:8 (MSC:PBMC).

Proliferation Assay

The culture was incubated with 1 μCi/mL 3H-thymidine for 24 hours beforeend of culture to measure the proliferation. Then, cells were washedtwice with ice-cold PBS and twice with ice-cold 5% trichloroacetic acid(TCA). Finally, cells were lysed with 0.1N NaOH-0.1% Triton-X100.Supernatants were collected and analyzed on a beta-counter in presenceof scintillation liquid. Results were done in cpm (“count perminute”=disintegration number of radioactive element observed in seriesof successive counts of one minute). IL-1β (beta) detection (By ELISA).Levels of IL-1β from PBMC or culture supernatants were measured byQuantikine ELISA kit (R&D Systems Inc, Mineapolis, USA). The minimumdetectable concentration of IL-1β was estimated at 1.0 pg/mL.

The release of IL-1beta by PBMC and the proliferation of PBMC, inducedby PHA stimulation, were set as controls, and compared with the measuresfor PBMC treated with HA and/or A2A.

The inhibition of PBMC release of IL-1beta and the inhibition of PBMCproliferation, induced by LPS stimulation of TH1 cells, were alsomeasured and provided similar trends as for PHA stimulated cells.

Effects of HA Alone

IL-1β detection Proliferation Conditions tested (pg/mL) (cpm) PBMC ND*769 PBMC + PHA 2185 16069 PBMC + HA 2095 364 PBMC + PHA + HA 6394 9087IL-1β detection Proliferation Conditions tested (%) (%) PBMC ND* 5PBMC + PHA 100 100 PBMC + HA 96 2.2 PBMC + PHA + HA 293 57 *notdetermined

IL-1beta secretion induced by PHA stimulation shows a significantincrease over basal condition (where IL-1beta levels were undetectable)and was set at 100. Proliferation of PMBC cells also significantlyincreased, by a factor of about 20. Surprisingly, a similar cytokineincrease (over basal condition) was observed with addition of HA alone,while PBMC proliferation was not affected. When stimulated by PHA, theaddition of HA synergistically increases IL-1beta secretion, 3 timesover the stimulated conditions, but was able to reduce PBMCproliferation by a factor 2.

Effects of A2A Alone

IL-1β detection Proliferation Conditions tested (pg/mL) (cpm) PBMC ND*769 PBMC + A2A ND* 786 PBMC + PHA 2185 16069 PBMC + PHA + A2A ND* 16212IL-1β detection Proliferation Conditions tested (%) (%) PBMC ND* 5PBMC + A2A ND* 4.9 PBMC + PHA 100 100 PBMC + PHA + A2A ND* 101 *notdetermined

A2A had no effect on basal (unstimulated) conditions. Interestingly,when stimulated by PHA, the addition of A2A totally suppressed theincrease in IL-1beta secretion due to PHA stimulation, to undetectablelevels, but was not able to inhibit PBMC proliferation.

Effects of Combination of A2A & HA

IL-1β detection Proliferation Conditions tested (pg/mL) (cpm) PBMC + PHA2185 16069 PBMC + PHA + HA 6394 9087 PBMC + PHA + HA + A2A 3053 5445IL-1β detection Proliferation Conditions tested (%) (%) PBMC + PHA 100100 PBMC + PHA + HA 292 57 PBMC + PHA + HA + A2A 139 34 *not determined

Interestingly, the combination HA/A2A demonstrates a potentanti-inflammatory effect over the PHA/HA condition. Indeed, the additionof A2A was able to significantly revert (by over 50%) the increase inIL-1beta secretion due to PHA/HA stimulation, bringing its level to theone of the PHA alone condition. Surprisingly, A2A was further able toinhibit PBMC proliferation, by another 35%.

Effects of MSC Addition

Testing the effects of MSC on both PBMC-proliferation and IL-1βsecretion, a stimulatory effect was observed over basal (unstimulated)conditions. Surprisingly, the proliferative effect was maintained instimulated conditions (e.g., presence of PHA), but instead of apro-inflammatory effect on IL-1β secretion, a potent inhibitory effectwas observed (−62%).

IL-1β detection Proliferation Conditions tested (pg/mL) (cpm) PBMC ND*769 PBMC + MSC 229 1612 PBMC + PHA 2185 16069 PBMC + PHA + MSC 841 26192IL-1β detection Proliferation Conditions tested (%) (%) PBMC ND* 100PBMC + MSC +++ 209 PBMC + PHA 100 100 PBMC + PHA + MSC 38 163 *notdetermined

In PHA stimulated conditions and in presence of hyaluronic acid, MSCswere modestly (about 10%) anti-inflammatory and anti-proliferative.

IL-1β detection Proliferation Conditions tested (pg/mL) (cpm) PBMC ND*769 PBMC + PHA 2185 16069 PBMC + PHA + HA 6394 9087 PBMC + PHA + HA +MSC 5769 8673 IL-1β detection Proliferation Conditions tested (%) (%)PBMC ND* 5 PBMC + PHA 100 100 PBMC + PHA + HA 292 57 PBMC + PHA + HA +MSC 264 54 *not determined

Finally in PHA stimulated conditions and in presence of A2A, MSCs had noinfluence.

IL-1β detection Proliferation Conditions tested (pg/mL) (cpm) PBMC ND*769 PBMC + PHA 2185 16069 PBMC + PHA + A2A ND  16212 PBMC + PHA + A2A +MSC  314 17061 IL-1β detection Proliferation Conditions tested (%) (%)PBMC ND* 5 PBMC + PHA 100 100 PBMC + PHA + A2A ND  100 PBMC + PHA +A2A + MSC  14 106 *not determined

During inflammation, lower molecular weight fragments of hyaluronan areknown to be inflammatory and immune-stimulatory agents by inducing thesecretion of cytokines such as IL-6 and monocyte chemoattractant protein(MCP-1). HA has also the property to enhance the adhesion of lymphocytesand monocytes to the extracellular matrix (Yamawaki et al., 2009). Thepresent experiments show that hyaluronic acid has a potent stimulatoryeffect on cytokine (e.g., IL-1β) production without PBMC proliferation.

It has been demonstrated that clonidine alters the Th1/Th2 cytokineproduction (Xu et al., 2007; Cook-Mills et al., 1998). The presentexperiments show that the addition of alpha 2 adrenoreceptor agoniststotally suppresses the increase in cytokine (e.g., IL-1beta) secretiondue to PHA stimulation, but is not able to inhibit PBMC proliferation.

Surprisingly, the combination HA/A2A demonstrates a potentanti-inflammatory effect both on cytokine production and inflammatorycell proliferation. Indeed, the addition of A2A to HA was able tosignificantly inhibit the increase in IL-1beta secretion and was furtherable to inhibit PBMC proliferation.

An in vitro property of MSCs is their ability to reduce theproliferation of lymphocytes of various types while enjoying immuneprivilege in vitro and in vivo (Bocelli-Tyndall et al., 2007). However,it was shown that the number of MSCs and the incubation time were keyfactors in MSCs' lymphocyte regulation: inhibition of lymphocyteproliferation was dose-dependent on MSC concentrations (LeBlanc in2003).

Interestingly, these results show that MSC have a moderate stimulatoryeffect on unstimulated PBMC by increasing both IL-1beta secretion andincrease PBMC proliferation. On the contrary, MSC displayed a potentanti-inflammatory effect (−62%) on IL-1beta secretion but increased PBMCproliferation. The inventors have hypothesized that MSCs did not induceproliferation of PBMC, but were able to increase their survival rate.This is illustrated by the attachment of PBMCs on the surface of MSCs(see enclosed FIG. 1). This seems to be confirmed by the increase inviability observed with PHA stimulated PBMCs in presence of MSCs; aviability increased by over 20%.

Regenerative Effects

MSCs were culture in chondrogenic medium for 7, 14 and 21 days and 4conditions were tested

-   -   control    -   A2A: 5 μMol aminoclonidine    -   HA: 200 μg/ml of hyaluronic acid    -   HA+A2A: 200 μg/ml of hyaluronic acid and 5 μMol aminoclonidine

At day 14 and 21, pellets were fixed in 10% buffered formalin for 6hours, and washed twice in PBS. Pellets were then embedded in OCT(Sakura Finetek, Belgium). Sections were cut at a thickness of 5 μm andwere stained with Alcian blue, before counterstained with Nuclear fastred (Klinipath, Belgium)

Alcian blue is one of the most widely used cationic dyes for thedemonstration of glycosaminoglycans (GAGs). GAGs will be stained blue,while Nuclear fast red will counterstain cells nuclei in pink or red,and cytoplasm in pale pink. In brief, the procedure is 1) Stain inalcian blue solution for 30 minutes; 3) Wash in running tap water for 2minutes; 4) Rinse in distilled water; 5) Counterstain in nuclear fastred solution for 5 minutes; 6) Wash in running tap water for 1 minute;7) Dehydrate through 95% alcohol, and 2 changes of absolute alcohol, 3minutes each; 8) clear in Ultraclear, a xylene substitute (Klinipath,Belgium); 9) Mount with Ultrakit mounting medium (Klinipaht, Belgium).Toluidine blue stains proteoglycan in red-purple (metachromaticstaining) and the nucleus in blue (orthochromatic staining).Metachromasia, tissue elements staining a different color from the dyesolution, is due to the pH, dye concentration and temperature of thebasic dye. Blue or violet dyes will show a red color shift, and red dyeswill show a yellow color shift with metachromatic tissue elements.Toluidine Blue is an often used to stain proteoglycans andglycosaminoglycans in tissues such as cartilage.

Results.

Although pellets from different conditions were of similar size (mean of0.7 mm; range from 0.9 to 0.4 mm), Alcian Blue staining reveals thatcellularity of pellets was significantly affected by the changes inconditions. For example, the addition of hyaluronic acid stronglyreduced the cellularity of the pellet. In parallel, matrix (i.e.,mucopolysaccharides and glycosaminoglycans) content was almost absent incontrol and A2A alone conditions as illustrated in FIG. 2 by the absenceof blue staining while in the hyaluronic acid and hyaluronicacid/clonidine conditions, the slides were uniformly blue stained,demonstrating an important matrix production. The combined conditionHA/A2A appears to further increase the matrix production over HA alonecondition.

FIG. 2: Alcian blue staining at low magnification power (Top) and highmagnification power (Bottom) for the different culture conditions:control, A2A (clonidine alone), HA (hyaluronic acid alone) and HA/A2A(combination of hyaluronic acid and clonidine)

Interestingly, cell nucleus staining demonstrate that the addition ofclonidine (HA/A2A) to hyaluronic acid (HA) increases the cellularity ofpellet.

In conclusion, the combination of hyaluronic acid, an anti-inflammatorycompound such as an alpha2 adrenoreceptor agonist and mesenchymalstromal cell displays surprisingly synergistic anti-inflammatory andregenerative effects:

-   -   The presence of mesenchymal stromal cells are required for the        de novo production of cartilage matrix;    -   This production is significantly increased (and accelerated) by        the addition of hyaluronic acid, but the addition of hyaluronic        acid has potent pro-inflammatory effects;    -   These hyaluronic acid potent pro-inflammatory effects are        totally neutralized by the addition of alpha2-adrenergic        agonists;    -   The addition of alpha2-adrenergic agonist agents supports the        regenerative effects of the combination of mesenchymal stromal        cells and hyaluronic acid by increasing the cellularity of the        cell preparation and the production of matrix.

REFERENCES

-   Bocelli-Tyndall et al. (Rheumatology; 46:403-408 (2007)).-   Bondeson et al. (Arthritis Res Ther; 8(6):R187 (2006)).-   Cook-Mills et al., 1995. (Immunology. 85 544-549 (1995)).-   Le Blanc et al., (Scandinavian Journal of Immunology, 57, 11-20    (2003)).-   Levesque et al., (The Journal of Immunology, 166: 188-196 (2001)).-   Mchugh et al. (Clin Exp Immunol. 99:160-167 (1995)).-   Pelletier et al. (Arthritis Rheum. 44(6):1237-47 (2001)).-   Yamawaki H et al. (Glycobiology, 19, 83-92 (2009)).

1.-14. (canceled)
 15. A method of treating a osteoarticular disease in amammalian subject, comprising administering to the subject apharmaceutical composition comprising: one or more glycosaminoglycan(s)an effective amount of mesenchymal stem cells an effective amount of ananti-inflammatory compound selected from the group consisting of analpha 2 adrenergic receptor agonist, a corticosteroid, a non-steroidalanti-inflammatory drug, an anti-TNF-alpha antibody; and wherein thepharmaceutical composition reduces joint degeneration in the subjectassociated with the osteoarticular disease thereby providing clinicalimprovement of the osteoarticular disease.
 16. The method according toclaim 15, wherein an injectable solution is administrated to thesubject.
 17. The method according to claim 15, wherein thepharmaceutical composition is administered to the subject throughintra-articular injection.
 18. The method according to claim 16, whereinthe injectable solution is administered to the subject twice a week. 19.The method according to claim 16, wherein the injectable solution isadministered to the subject once a week.
 20. The method according toclaim 16, wherein the injectable solution is administered to the subjectonce in two or three weeks or less.
 21. The method according to claim15, which comprises administering an amount of the pharmaceuticalcomposition such that the subject receives from about 0.1 to about 100mg/kg of body weight of anti-inflammatory compound.
 22. The methodaccording to claim 15, which comprises administering an amount of thepharmaceutical composition such that the subject receives from about 0.1to about 100 mg/kg of body weight of the glycosaminoglycan.
 23. Themethod according to claim 15, wherein the mesenchymal stem cells areobtained from bone marrow concentrate.
 24. The method according to claim15, wherein the osteoarticular disease is osteoarthritis, degenerativearthritis, gonarthrosis, coxarthrosis, gout, chondrocalcinosis, calciumhydroxyapatite crystal deposition disease, spondyloarthropathies,ankylosing spondylitis, Reiter's Syndrome, psoriatic arthropathy,enteropathic arthritis, rheumatoid arthritis, neuropathic arthropathy,or acute rheumatic fever.
 25. The method of claim 15, wherein thesubject is a human.
 26. The method of claim 15, wherein thepharmaceutical composition further comprises a pharmaceutical adequatecarrier or diluent.